Fundamental question in developmental hematopoiesis is to understand how the hematopoietic system is established in the developing embryo. As hematopoietic and endothelial cells develop closely together in space and time, it has been postulated that they share a common progenitor, the hemangioblast. For the past funding period (1996-present), we have demonstrated that Flk-1 expressing blast colony forming cells (BL CFCs) represent the hemangioblast, a common progenitor of hematopoietic and endothelial cells. However, as our previous studies have utilized an in vitro differentiation model system of embryonic stem (ES) cells, it still remains to be verified whether BL-CFCs truly establish hematopoietic and endothelial cells in the developing embryo. Thus, this proposal seeks to determine in vivo developmental potential of Flk-1 cells. Specifically, in vivo as well as in vitro generated Flk-1+ cells will be injected into blastocysts, neonate, and adult mice and examined if donor derived hematopoietic and endothelial cells can be found in the recipients. Determining their in vivo developmental potential will enhance our understandings of the establishment of the hematopoietic system and our ability to utilize hemangioblast/Flk-1+ cells in the future for cell-based transplantation and gene therapy. With regard to the initial hematopoietic site, recent studies suggest that hematopoietic cells also develop within the para-aortic-splanchnopleura (PAS)/aorta-gonad-mesonephros (AGM) of the developing embryo. As the relationship between the yolk sac and AGM hematopoiesis is unknown, this proposal seeks to determine the progenitor relationship between the yolk sac and intraembryonic hematopoiesis. Specifically, we plan to mark individual Flk-1+ cells with unique molecular markers/tags via retroviral infection. Marked Flk-1+ cells will be injected into blastocysts, and yolk sac and AGM will be analyzed for molecular markers at different time points. Determining the relationship between the yolk sac and intra-embryonic hematopoiesis will be a step forward in developmental hematopoiesis, as an initial pathway of the hematopoietic progenitor migration is unknown. Finally, this proposal seeks to determine molecular mechanisms involved in the hemangioblast development. Specifically, genes uniquely expressed in Flk-1+ cell compartments are to be isolated and characterized. Better understanding of genes expressed in the hemangioblast cell population should further our understandings of the hemangioblast, hematopoietic, and endothelial cell lineage development.